CN110716052B - Kit for detecting human eosinophil cationic protein and myeloperoxidase - Google Patents
Kit for detecting human eosinophil cationic protein and myeloperoxidase Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a detection kit for human eosinophil cationic protein and myeloperoxidase and application thereof. The two kits of the invention adopt a reaction mode of a double-antibody sandwich one-step method, and can simultaneously carry out very specific quantitative and qualitative detection on the human eosinophil cationic protein and the myeloperoxidase molecule in serum and nasal secretion samples of patients. The method not only effectively utilizes the principle of chemiluminescence technology, but also applies enzyme-catalyzed luminous substrates on the basis of enzyme-linked immunoassay, improves the detection sensitivity, has the advantages of high stability, good specificity, good accuracy, simple operation and no radioactive pollution, and can provide more specific, rapid and reliable basis for clinical diagnosis of inflammatory diseases such as bronchial asthma, nasosinusitis, rhinitis and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a detection kit for human eosinophil cationic protein and myeloperoxidase and application thereof.
Background
Allergic rhinitis (allergic rhinitis, AR) and chronic sinusitis (chronic rhinosinusitis, CRS) are the most common diseases of ear, nose, throat and neck surgery. The diagnosis often depends on the symptoms and signs of the patient. The cytological examination of nasal secretions is used as an important auxiliary diagnosis method, is beneficial to the accurate diagnosis and the personalized treatment of rhinitis patients, has complicated operation and lacks unified standards, and is not widely applied in clinic although being developed. In our previous cytological study of nasal secretions, it was determined that eosinophils and neutrophils account for the majority (> 98%) of nasal secretions, with AR patients 'nasal secretions mostly characterized by eosinophils and CRS patients' nasal secretions by neutrophils. Because the sampling process of the nasal cytology examination has a certain instability, and can be influenced and restricted by the condition of the patient at the time of the visit (such as too little secretion amount, patient mismatch, etc.), the accuracy of the result is affected. In contrast, nasal secretion is more convenient and stable to obtain. Eosinophil Cationic Protein (ECP) is an alkaline protein, an important index reflecting the activation amount of eosinophils, and is one of the clinical signs of allergic patients. Myeloperoxidase (MPO) is a heme protease containing heme prosthetic groups, and is also the most abundant pro-inflammatory enzyme stored in neutrophils, reflecting neutrophil activation.
At present, no product for judging the inflammatory property of the nasal cavity exists clinically, and no bid product exists, even no product exists. The judgment of the nasal inflammation and the adjustment of the treatment by doctors are completely carried out by experience. ECP and MPO have wide and important application value in clinic; the nasal cavity local inflammation is accurately judged, so that the accurate treatment of rhinitis is guided, and the treatment effect is objectively evaluated.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art and providing a detection kit for human eosinophil cationic protein and myeloperoxidase and application thereof.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
a human eosinophil cationic protein and myeloperoxidase assay kit comprising the following two types:
a) A chemiluminescent immunoassay kit (CLIA) and a chemiluminescent immunoassay kit (ELISA) of human eosinophil cationic protein and myeloperoxidase, comprising the components of:
(1) a human eosinophil cationic protein and a human myeloperoxidase standard;
(2) a carrier coated with a human eosinophil cationic protein monoclonal antibody and a human myeloperoxidase monoclonal antibody;
(3) a labeled human eosinophil cationic protein monoclonal antibody and a human myeloperoxidase monoclonal antibody; (3) the labeled antibody and the coated antibody in the step (2) are paired antibodies, and form a double-antibody sandwich structure with the human eosinophil cationic protein and the human myeloperoxidase respectively; no cross reaction exists between the human eosinophil cationic protein pairing antibody and the human myeloperoxidase pairing antibody;
(4) a substrate for color development;
b) An immunochromatographic assay kit (colloidal gold) for human eosinophil cationic protein and myeloperoxidase, comprising:
(1) a sample loading pad stuck on one end of the bottom plate;
(2) a colloidal gold pad containing a labeled human eosinophil cationic protein monoclonal antibody and a human myeloperoxidase monoclonal antibody in intimate pressure contact with the loading pad;
(3) the nitrocellulose NC film is tightly pressed with one end of the colloidal gold pad, a detection line T and a quality control line C which are mutually separated are coated on the nitrocellulose NC film, a human eosinophil cationic protein monoclonal antibody and a human myeloperoxidase monoclonal antibody which are matched with the labeled antibody (2) are coated on the detection line T, and a goat anti-mouse IgG antibody is coated on the quality control line C; the coated antibody on the detection line T and the labeled antibody in the step (2) are paired antibodies, and form a double-antibody sandwich structure with the human eosinophil cationic protein and the human myeloperoxidase respectively; no cross reaction exists between the human eosinophil cationic protein pairing antibody and the human myeloperoxidase pairing antibody;
(4) a sample pad tightly pressed with the nitrocellulose NC film.
In the above scheme, the preparation method of the vector coated with the human eosinophil cationic protein monoclonal antibody and the human myeloperoxidase monoclonal antibody in the A) comprises the following steps: and taking carbonate buffer solution as a solvent, uniformly mixing the human eosinophil cationic protein monoclonal antibody and the human myeloperoxidase monoclonal antibody with the solvent, loading the mixture on a carrier, cleaning the carrier, sealing the carrier by adopting sealing liquid, and drying the carrier to obtain the carrier coated with the human eosinophil cationic protein monoclonal antibody and the human myeloperoxidase monoclonal antibody.
In the above scheme, the carrier is a microplate, magnetic particles, plastic tube or plastic beads.
In the above scheme, the enzyme used for labeling in A) is alkaline phosphatase or horseradish peroxidase.
In the above scheme, the chromogenic substrate in the chemiluminescent immunoassay kit in A) is luminol, isoluminol, (adamantane) -1, 2-dioxyethane, 3- (2 '-helical adamantane) -4-methoxy-4- (3' -phosphoryloxy) phenyl-1, 2-dioxyethane, CSPD or CDP-Star; the chromogenic substrate in the chemochromic immunoassay kit is 3,3', 5' -tetramethyl benzidine or diaminobenzidine.
In the above scheme, the preparation process of the colloidal gold pad in B) is as follows: adding labeled human eosinophil cationic protein monoclonal antibody and human myeloperoxidase monoclonal antibody into the colloidal gold solution by a chloroauric acid-trisodium citrate reduction method, stirring for 2 hours at room temperature, adding bovine serum albumin with the final concentration of 1 percent, sealing with polyethylene glycol 20000 for 20 minutes, centrifuging, discarding the supernatant, redissolving with a colloidal gold working solution, and uniformly paving the solution on a glass fiber membrane or a non-woven fabric to prepare the colloidal gold pad.
The eosinophil cationic protein and myeloperoxidase detection kit is applied to synchronously detecting eosinophil cationic protein and myeloperoxidase products.
The invention has the beneficial effects that: the two kits of the invention adopt a reaction mode of a double-antibody sandwich one-step method, and can simultaneously carry out very specific quantitative and qualitative detection on the human eosinophil cationic protein and the myeloperoxidase molecule in serum and nasal secretion samples of patients. The chemiluminescence immunoassay kit (CLIA) and the chemiluminescence immunoassay kit (ELISA) of the human eosinophil cationic protein and the myeloperoxidase not only effectively utilize the principle of chemiluminescence technology, but also use enzyme-catalyzed luminous substrates on the basis of enzyme-linked immunoassay, thereby improving the sensitivity of detection, and simultaneously having the advantages of high stability, good specificity, good accuracy, simple operation and no radioactive pollution. The immunochromatography detection kit (colloidal gold) for the cationic protein and the myeloperoxidase of the human eosinophil has accurate, stable and reliable detection result; is particularly suitable for the health requirements of the masses in the basic level and rural areas, and can provide more specific, rapid and reliable basis for the clinical diagnosis of inflammatory diseases such as bronchial asthma, nasosinusitis, rhinitis and the like.
Drawings
FIG. 1 is a graph showing the detection results of the immunochromatographic assay kit (colloidal gold) for human eosinophil cationic protein and myeloperoxidase described in example 2.
FIG. 2 is a linear plot of standard samples in the kit prepared in example 3.
Fig. 3 shows correlation results of detection results of chemiluminescent immunoassay kit (CLIA) of human eosinophil cationic protein and myeloperoxidase of the present invention and ECP immunofluorescence assay kit of Thermo Fisher Scientific company in us, and shows that the correlation results reach r= 0.9757 (P < 0.0001) when the content of ECP in30 samples is detected.
Detailed Description
For a better understanding of the present invention, the following examples are further illustrated, but are not limited to the following examples.
Example 1
A human eosinophil cationic protein and myeloperoxidase chemiluminescent immunoassay kit (CLIA) and a chemiluminescent immunoassay kit (ELISA), comprising the following components:
1) Human eosinophil cationic protein and myeloperoxidase protein standard;
2) Microplates coated with monoclonal antibodies to human eosinophil cationic protein and monoclonal antibodies to human myeloperoxidase;
3) Horseradish peroxidase-labeled human eosinophil cationic protein monoclonal antibodies and myeloperoxidase monoclonal antibodies;
4) Chemiluminescent substrate AMPPD acted by the above enzyme;
wherein:
1. the human eosinophil cationic protein and myeloperoxidase standard was prepared by the following method: phosphate buffer solution with the pH value of 7.4 and fetal bovine serum are mixed according to the volume ratio of 4:1 to prepare a basic buffer solution, and the basic buffer solution is used for respectively diluting the human eosinophil cationic protein and the myeloperoxidase to the concentration of 0, 25, 64, 160, 400 and 1000ng/mL.
2. The preparation method of the microplate coated with the human eosinophil cationic protein monoclonal antibody and the myeloperoxidase monoclonal antibody comprises the following steps:
1) Mixing 50mM carbonate buffer solution with pH value of 9.6 as solvent with human eosinophil cationic protein monoclonal antibody and myeloperoxidase monoclonal antibody to prepare mixed solution with concentration of 5 mug/mL, adding into each hole of the micro-porous plate, and standing at 4 ℃ for 24h with 120 mug/hole;
2) Washing the microwell plate 3 times with PBS at pH7.4, 300 μl each time;
3) Adding 300 mu L of sealing liquid with the pH value of 7.3 into each cleaned microplate, standing overnight at 4 ℃, throwing away the sealing liquid, beating to dry on absorbent paper, immediately vacuum-packaging after dehumidifying and drying at room temperature for 24 hours, and storing at 4 ℃;
the sealing liquid is prepared by the following method: 8g NaCl, 1.43g anhydrous Na was taken 2 HPO 4 0.24g anhydrous KH 2 PO 4 0.2g KCl, 10g BSA, 25g sucrose and 0.5mL Proclin300 were added deionized water to 1000mL.
3. Alkaline phosphatase-labeled monoclonal antibodies to human eosinophil cationic protein and myeloperoxidase
Coupling the monoclonal antibody of the human eosinophil cationic protein and the monoclonal antibody of the myeloperoxidase with horseradish peroxidase by a sodium periodate method, fully dialyzing by a PBS buffer solution with the pH value of 7.3, adding equal volume of glycerol into the dialyzed conjugate solution, diluting by a 20% bovine serum enzyme diluent until the concentration of the enzyme-labeled antibody is 1:10000 which is below 20 ℃ below minus 20 ℃, and preserving;
the formula of the 20% bovine serum enzyme diluent is as follows:
4. chemiluminescent substrate 3- (2 '-spiral adamantane) -4-methoxy-4- (3' -phosphoryloxy) phenyl-1, 2-dioxyethane (AMPPD) acted by the above enzyme
The formula of the AMPPD chemiluminescent substrate is as follows:
5. semi-finished product and finished product composition
The product obtained by the steps is split-packed to obtain a semi-finished product. Three parts are randomly extracted for specificity, precision, sensitivity and stability test, and the kit is assembled into a chemiluminescence immunoassay kit (CLIA) and a chemochromogenic immunoassay kit (ELISA) after the kit is qualified.
Experiments prove that: the corresponding human eosinophil cationic protein and myeloperoxidase chemiluminescent immunoassay kit (CLIA) and chemochromogenic immunoassay kit (ELISA) can be prepared by substituting plastic tubes for the microwell plates of example 1, step 2) with example 1.
The magnetic particles or plastic beads are used for replacing the micro-pore plates in the step 2) of the embodiment 1, blank micro-pore plates or plastic tubes are put into the kit, and corresponding human eosinophil cationic protein and myeloperoxidase chemiluminescent immunoassay kit (CLIA) and chemochromogenic immunoassay kit (ELISA) can be prepared in other embodiments 1;
experiments prove that: the corresponding human eosinophil cationic protein and myeloperoxidase chemiluminescent immunoassay kit (CLIA) and chemiluminescent immunoassay kit (ELISA) can be prepared by substituting PBS at pH 7.3 or 7.5, or PBST at pH 7.3, 7.4 or 7.5 for PBS at pH7.4 of step 2 of example 1, otherwise as in example 1;
experiments prove that: the blocking solution with the pH value of 7.2, 7.4 or 7.5 is used for replacing the blocking solution with the pH value of 7.3 in the step 2 in the embodiment 1, and corresponding human eosinophil cationic protein and myeloperoxidase chemiluminescent immunoassay kit (CLIA) and chemochromogenic immunoassay kit (ELISA) can be prepared in the same manner as in the embodiment 1;
experiments prove that: the corresponding human eosinophil cationic protein and myeloperoxidase chemiluminescent immunoassay kit (CLIA) and chemochromogenic immunoassay kit (ELISA) can be prepared by substituting (adamantane) -1, 2-dioxyethane, CSPD or CDP-Star for 3- (2 '-helical adamantane) -4-methoxy-4- (3' -phosphoryloxy) phenyl-1, 2-dioxyethane of step 4 of example 1, otherwise as in example 1;
experiments prove that: the corresponding human eosinophil cationic protein and myeloperoxidase chemiluminescent immunoassay kit (CLIA) and chemiluminescent immunoassay kit (ELISA) can be prepared in the same manner as in example 1, except that the concentrated wash of pH 7.2, 7.4 or 7.5 is used instead of the concentrated wash of pH 7.3 in step 5 of example 1.
Pairing antibody interaction: to avoid non-specific binding between two pairs of paired antibodies, interactions between paired antibodies need to be excluded, and the experimental protocol was designed as follows:
A. the antigen-antibody cross reaction: mixing 50mM carbonate buffer solution with pH value of 9.6 as solvent with human ECP (eosinophil cationic protein), coating an ELISA plate with mixed solution with concentration of 5 mug/mL, and detecting with a pair of MPO antibodies respectively, wherein the result is negative, and the MPO antibody can not identify human ECP (eosinophil cationic protein);
mixing 50mM carbonate buffer solution with pH value of 9.6 as solvent with human MPO (myeloperoxidase), coating the enzyme label plate with mixed solution with concentration of 5 μg/mL, and detecting with a pair of ECP antibodies respectively, wherein the ECP antibodies can not recognize human MPO (myeloperoxidase) as negative result;
B. the presence or absence of cross-reaction between antibody-antibody: mixing 50mM carbonate buffer solution with pH value of 9.6 as solvent with ECP (eosinophil cationic protein) and MPO (myeloperoxidase) monoclonal antibody to prepare mixed solution coated ELISA plate with concentration of 5 μg/mL; meanwhile, 50mM carbonate buffer solution with pH value of 9.6 is adopted as solvent to be mixed with ECP (eosinophil cationic protein) monoclonal antibody to prepare mixed solution with concentration of 5 mug/mL for coating the ELISA plate; the ECP proteins with the same concentration gradient were used for detection, and the results showed that: the detection results of the single antibody and the two antibodies are consistent, which shows that the MPO (myeloperoxidase) monoclonal antibody has no influence on the detection result of ECP;
mixing 50mM carbonate buffer solution with pH value of 9.6 as solvent with ECP (eosinophil cationic protein) and MPO (myeloperoxidase) monoclonal antibody to prepare mixed solution coated ELISA plate with concentration of 5 μg/mL; simultaneously, 50mM carbonate buffer solution with the pH value of 9.6 is adopted as a solvent to be mixed with MPO (myeloperoxidase) monoclonal antibody to prepare a mixed solution with the concentration of 5 mug/mL to coat the ELISA plate; the detection with the same concentration gradient of MPO (myeloperoxidase) showed: the detection results of the single antibody and the two antibodies are consistent, which shows that the ECP (eosinophil cationic protein) monoclonal antibody has no influence on the detection result of MPO.
A method for detecting human eosinophil cationic protein and myeloperoxidase using the human eosinophil cationic protein and myeloperoxidase chemiluminescent immunoassay kit (CLIA) and the chemiluminescent immunoassay kit (ELISA), comprising the steps of:
(1) Sample adding: respectively arranging a standard hole, a sample hole to be detected and a blank hole; setting a standard hole 7 holes, sequentially adding 100 mu L of standard substance, adding 100 mu L of sample diluent into a blank hole, adding 100 mu L of sample to be measured Yu Kongjia, and incubating an ELISA plate for 30 minutes at 37 ℃;
(2) Discarding the liquid, spin-drying without washing;
(3) 100 mu L of enzyme-labeled antibody working solution (prepared before use) is added into each hole, and the ELISA plate is incubated for 30 minutes at 37 ℃;
(4) Discarding the liquid in the holes, washing each hole with 250 mu L of washing liquid, soaking for 1 minute, sucking off (not touching the wall of the plate) or throwing off the liquid in the ELISA plate, laying a plurality of layers of absorbent paper on the experiment table, beating the ELISA plate downwards for several times (beating the liquid in the hole for drying) forcefully, repeatedly washing the plate for 3 times, and completely spin-drying the washing liquid in the hole after the last washing; automatic plate washing machine is also used.
(5) 100 mu L of biotin-labeled streptavidin working solution (prepared just before use) was added to each well, and incubated at 37℃for 30 minutes;
(6) Discarding the liquid in the holes, spin-drying, washing the plate for 5 times, and the method is the same as that in the step 4;
(7) Adding 100 mu L of substrate solution into each hole, and developing the color of the ELISA plate at 37 ℃ in a dark place (the reaction time is controlled to be 10-15 minutes and not more than 20 minutes, and the ELISA plate can be stopped when the front 3-4 holes of the standard holes have obvious gradient blue and the gradient of the rear 3-4 holes is not obvious);
(8) Adding 50 mu L of stop solution into each hole to stop the reaction, and turning blue to yellow immediately; the adding sequence of the stopping solution is the same as the adding sequence of the substrate solution as much as possible, if the color is uneven, the enzyme label plate is gently rocked to mix the solutions uniformly;
(9) After ensuring that there are no water drops at the bottom of the microplate and no bubbles in the wells, the optical density (o.d. value) of each well was measured immediately with a microplate reader at a wavelength of 450 nm.
Example 2
An immunochromatography detection kit (colloidal gold) for human eosinophil cationic protein and myeloperoxidase, the preparation method is as follows:
1. preparing a gold mark pad:
colloidal gold solution with diameter of 30-40 nm is prepared by a reduction method of human eosinophil cationic protein and myeloperoxidase antibody colloidal gold labeled chloroauric acid-trisodium citrate, and three parts of colloidal gold are taken after the preparation is finished and respectively used for 0.2M K 2 CO 3 Adjusting the solution to pH7.5, pH8.0 and pH8.5; then the solution is placed on a magnetic stirrer to be slowly stirred, 0.5mg, 1mg and 1.5mg of the labeled human eosinophil cationic protein and myeloperoxidase antibody are added into each 100ml of the solution, the solution is slowly dripped into a colloidal gold solution, the stirring is continued for 2 hours, then the solution is added into PEG2000 with the final concentration of 1% and BSA with the final concentration of 1% to be blocked for 20 minutes, the centrifugation is carried out at 12000r/min after the end of the labeling, the supernatant is discarded, and the precipitate is redissolved into colloidal gold working solutions with different proportions according to 50% of the original volume (pH 8.0, BSA is contained, sheep serum)Sucrose and surfactant). Then the marked colloidal gold solution is spread for 20cm according to 1ml solution 2 The mixture is added on a glass fiber film or a non-woven fabric, and the mixture is dried for 2 to 5 hours in a drying room with the temperature of 20 to 25 ℃ and the relative humidity of less than 30 percent to prepare the colloidal gold pad.
2. Coating film preparation:
NC membrane coating, diluting the coated human eosinophil cationic protein and myeloperoxidase antibody to 0.5mg/ml, 1mg/ml and 1.5mg/ml by using PBS (phosphate buffer solution) with pH7.4 with 0.01M, respectively diluting goat anti-mouse IgG to 1mg/ml and 2mg/ml, respectively coating the NC membrane by scribing according to 1.2ul/cm by using a membrane spraying instrument, and drying the NC membrane for 2-5 hours at the temperature of 20-25 ℃ and the relative humidity of less than 30% in a drying room after the coating is completed.
3. And (3) assembling a test paper card:
placing a coated NC film in the middle of a plastic bottom plate for pasting, cutting a colloidal gold pad into a proper width, overlapping the colloidal gold pad on one side of a NC film T line, overlapping 1/4 of the colloidal gold pad for pasting, overlapping and pasting a sample pad on the other side of the colloidal gold pad, overlapping 1/3 of the colloidal gold pad for pasting in a drying chamber with the temperature of 20-25 ℃ and the humidity of less than 40%; the sample absorbing pad is lapped on one side of the NC film C line, and 1/10 of the sample absorbing pad is stuck; finally, cutting the stuck plastic plate into test strips with the width of 3-5 mm by a cutting machine, and then putting the test strips into a plastic card to form the test paper card.
Pairing antibody interaction: to avoid non-specific binding between two pairs of paired antibodies, interactions between paired antibodies need to be excluded, and the experimental protocol was designed as follows:
A. the antigen-antibody cross reaction: NC film coating human ECP (eosinophil cationic protein) was diluted to 0.5mg/ml, 1mg/ml, 1.5mg/ml with 0.01M PBS pH7.4, then streaked coating was performed on NC film with a film spray machine at 1.2ul/cm, and NC film was dried for 2-5 hours at 20-25℃in a drying room with a relative humidity of < 30%. Detecting with a pair of MPO antibodies respectively, wherein the result is negative, and the MPO antibodies cannot recognize human ECP (eosinophil positive protein);
NC film coating human MPO (myeloperoxidase) was diluted to 0.5mg/ml, 1mg/ml, 1.5mg/ml with 0.01M PBS pH7.4, then streaked coating was performed on NC film at 1.2ul/cm with a film spray apparatus, and NC film was dried at 20-25℃for 2-5 hours in a drying room with a relative humidity of < 30% after coating was completed. Detecting with a pair of ECP antibodies respectively, wherein the result is negative, and the ECP antibodies cannot recognize human MPO (myeloperoxidase);
B. the presence or absence of cross-reaction between antibody-antibody: NC membrane coating, diluting ECP (eosinophil cationic protein) and MPO (myeloperoxidase) monoclonal antibodies to 0.5mg/ml, 1mg/ml and 1.5mg/ml by using 0.01M PBS (phosphate buffer solution) pH7.4, respectively streaking and coating the NC membrane by using a membrane spraying instrument according to 1.2ul/cm, and drying the NC membrane for 2-5 hours at the temperature of 20-25 ℃ and the relative humidity of less than 30% in a drying room after coating is completed; coating ECP (eosinophil cationic protein) monoclonal antibody by the same process; the ECP proteins with the same concentration gradient were used for detection, and the results showed that: the detection results of the single antibody and the two antibodies are consistent, which shows that the MPO (myeloperoxidase) monoclonal antibody has no influence on the detection result of ECP;
NC membrane coating, diluting ECP (eosinophil cationic protein) and MPO (myeloperoxidase) monoclonal antibodies to 0.5mg/ml, 1mg/ml and 1.5mg/ml by using 0.01M PBS (phosphate buffer solution) pH7.4, respectively streaking and coating the NC membrane by using a membrane spraying instrument according to 1.2ul/cm, and drying the NC membrane for 2-5 hours at the temperature of 20-25 ℃ and the relative humidity of less than 30% in a drying room after coating is completed; coating MPO (myeloperoxidase) monoclonal antibody by the same process; the detection with the same concentration gradient of MPO (myeloperoxidase) showed: the detection results of the single antibody and the two antibodies are consistent, which shows that the ECP (eosinophil cationic protein) monoclonal antibody has no influence on the detection result of MPO.
The detection method for detecting the human eosinophil cationic protein and the myeloperoxidase by using the immunochromatography detection kit (colloidal gold) for the human eosinophil cationic protein and the myeloperoxidase comprises the following steps:
(1) Sampling: the sampling paper strip can be gently placed into the nasal cavity (for example, nasal secretion is more, and the nasal secretion can be directly dipped). After secretion permeates the red line of the paper strip, the part above the red line can be sheared by a small scissors, and put into a secretion soaking bottle, and mixed and shaken for about 10 seconds;
(2) Sample adding: taking a test pen card, opening a cover in front of the soaking bottle, and dripping 10 soaking liquid drops into a hole groove at the lower end of the pen card;
(3) Color development: the test pencil card is vertically placed, so that the upward infiltration of liquid can be seen, and red color appears; reading after waiting for 15-20 minutes;
(4) Interpretation of the results: reading the mark: the control line (C) is the uppermost one, the MPO (M) is the middle one, and the ECP (E) is the lowermost one
1) Result 1 (as shown in fig. 1 (a)): if all three lines appear (C, M, E appears), it can be judged that the infectivity is dominant and the anti-infection treatment can be dominant in treatment;
2) Result 1 (as shown in fig. 1 (b)): if only the upper and lower lines are displayed (C and E are displayed), the allergic reaction is judged to be dominant, and the antiallergic treatment can be performed mainly on the basis of the treatment;
3) Result 1 (as shown in fig. 1 (c)): if only the first C line appears, normal person detection shows that the result is also available for other part of rhinitis patients with non-infectious and non-allergic symptoms, and the analysis, judgment and treatment are needed according to the specific symptoms.
Note that: (1) the other display results except for the 3 results are incorrect and need to be detected again.
(2) Judging the degree: for developing more than C-line as M or E deeper is "+++", the equivalent of the C line is "++", the weaker than C line is "+", and the non-developed is "-".
EXAMPLE 3 methodological significance of the kit of the invention
The ECP content in nasal secretions cannot be used as an independent index for diagnosing AR, but combining the expression of ECP and MPO in nasal secretions can well distinguish AR from CRS. The study is intended to diagnose nasal inflammation more accurately by analyzing inflammatory factors in nasal secretions, and becomes a more suitable clinical diagnosis method than nasal secretion cytology. Meanwhile, the expression of inflammatory factors in the AR group and the CRS group is obviously higher than that in a healthy control group, and the ECP and MPO levels are reduced and are close to normal after treatment, so that the anti-inflammatory agent is expected to become an objective index in the treatment of rhinitis and nasosinusitis.
As shown in fig. 2 (a), ECP concentrations in nasal secretions were significantly elevated in AR and CRS groups compared to CONTROL group (AR v.s CONTROL p= 0.0008;AR v.s CRS P<0.0001), and CRS group was significantly higher than AR group (p=0.0056); MPO concentrations in nasal secretions were significantly elevated in CRS groups compared to AR and control groups (both P < 0.0001), and AR groups were slightly higher than control groups (p= 0.3105).
As shown in fig. 2 (b), the eosinophil percentage in the AR group cytology was significantly higher than that in the CRS group and control group (P < 0.0001), with the CRS group slightly higher than the control group (p= 0.0493); the percentage of neutrophils in the cytological examination of the CRS group was significantly higher than that of the AR group and the control group (P < 0.0001), and the AR group was slightly higher than that of the control group (p=0.0163). (see FIG. 2).
Example 4
Clinical application of the chemiluminescent immunoassay kit (CLIA) of human eosinophil cationic protein and myeloperoxidase of the present invention was compared with ECP immunofluorescence assay kit of Thermo Fisher Scientific company in us:
the correlation between the kit prepared in example 1 of the present invention and the ECP immunofluorescence assay kit of Thermo Fisher Scientific company in the United states was compared with each other by simultaneously detecting 32 patients with allergic rhinitis and 30 patients with chronic sinusitis.
1. Sources and inclusion criteria for clinical serum specimens:
32 patients with allergic rhinitis, 18 men, 14 women, age 4-41 years, average (19.9±10.8) years, and standard reference allergic rhinitis diagnosis and treatment guidelines (2015, tianjin) were incorporated: (1) symptoms: the symptoms such as sneeze, watery nasal discharge, nasal itching, nasal obstruction and the like appear for 2 or more, and the symptoms are continuous or accumulated for more than 1 hour every day, and can be accompanied with eye symptoms such as eye itching, lacrimation, eye redness and the like; (2) the physical sign is as follows: pale and edema of nasal mucosa and nasal cavity water-like secretion are common; (3) allergen detection: at least one allergen SPT and/or serum specific IgE positive.
30 chronic rhinosinusitis patients, 16 men, 13 women, age 3-28 years, average (9.2±7.3) years, inclusion criteria reference chronic rhinosinusitis diagnosis and treatment guidelines (2012, kunming): (1) symptoms: one of nasal obstruction, viscosity or mucopurulent nasal discharge can be combined with headache, facial distending pain, hyposmia or loss; (2) nasal endoscopy: viscous or mucopurulent secretions from the internal nasal passages, olfactory clefts, nasal mucosa engorgement, oedema, without merging polyps; (3) imaging examination: the sinus CT scan reveals the sinus oronasal airway complex and/or the sinus mucositis lesions. In order to reduce allergic factor interference, the sinusitis selected in the study did not incorporate allergic rhinitis.
21 normal individuals, 2 men, 19 women, ages 32-60, average (38.3+ -5.6) years, incorporating the standard: no history of rhinitis has been available and respiratory diseases have not been developed recently.
The experiment was approved by the ethical committee of the university of science and technology in China.
2. The nasal secretion samples were each tested using the kit of example 1 of the present invention and the ECP immunofluorescence assay kit of US Thermo Fisher Scientific (operating according to the instructions), and the results were subjected to statistical analysis, indicating that the results of the two methods were highly correlated, as shown in FIG. 3.
3. Results
Comparative data from the detection of identical serum samples using the chemiluminescent immunoassay kit (CLIA) for human eosinophil cationic protein and myeloperoxidase and the ECP immunofluorescence assay kit from us Thermo Fisher Scientific company show that the two methods are highly correlated with a correlation coefficient r > 0.98 and a p value less than 0.01. The two methods are described as having equal use value.
It is apparent that the above examples are only examples given for clarity of illustration and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. And thus obvious variations or modifications to the disclosure are within the scope of the invention.
Claims (4)
1. A kit for detecting human eosinophil cationic protein and myeloperoxidase, comprising the following two types:
a) A chemiluminescent immunoassay kit and a chemiluminescent immunoassay kit for human eosinophil cationic protein and myeloperoxidase, which comprise the following components:
(1) a human eosinophil cationic protein and a human myeloperoxidase standard;
(2) the preparation method of the carrier coated with the human eosinophil cationic protein monoclonal antibody and the human myeloperoxidase monoclonal antibody comprises the following steps: taking carbonate buffer solution as a solvent, uniformly mixing a human eosinophil cationic protein monoclonal antibody and a human myeloperoxidase monoclonal antibody with the solvent, loading the mixture on a carrier, cleaning the carrier, sealing the carrier by using sealing liquid, and drying the carrier to obtain the carrier coated with the human eosinophil cationic protein monoclonal antibody and the human myeloperoxidase monoclonal antibody;
(3) a labeled human eosinophil cationic protein monoclonal antibody and a human myeloperoxidase monoclonal antibody; (3) the labeled antibody and the coated antibody in the step (2) are paired antibodies, and form a double-antibody sandwich structure with the human eosinophil cationic protein and the human myeloperoxidase respectively; no cross reaction exists between the human eosinophil cationic protein pairing antibody and the human myeloperoxidase pairing antibody;
(4) a substrate for color development;
b) A colloidal gold immunochromatography detection kit for human eosinophil cationic protein and myeloperoxidase comprises:
(1) a sample loading pad stuck on one end of the bottom plate;
(2) a colloidal gold pad containing a labeled human eosinophil cationic protein monoclonal antibody and a human myeloperoxidase monoclonal antibody, which is tightly pressed with a sample pad, wherein the preparation process of the colloidal gold pad comprises the following steps: preparing a colloidal gold solution by a chloroauric acid-trisodium citrate reduction method, adding a marked human eosinophil cationic protein monoclonal antibody and a human myeloperoxidase monoclonal antibody into the colloidal gold solution, stirring for 2 hours at room temperature, adding bovine serum albumin with the final concentration of 1 percent and polyethylene glycol 20000, sealing for 20 minutes, centrifuging, discarding the supernatant, redissolving with a colloidal gold working solution, and uniformly paving the solution on a glass fiber membrane or a non-woven fabric to prepare a colloidal gold pad;
(3) the nitrocellulose NC film is tightly pressed with one end of the colloidal gold pad, a detection line T and a quality control line C which are mutually separated are coated on the nitrocellulose NC film, a human eosinophil cationic protein monoclonal antibody and a human myeloperoxidase monoclonal antibody which are matched with the labeled antibody (2) are coated on the detection line T, and a goat anti-mouse IgG antibody is coated on the quality control line C; the coated antibody on the detection line T and the labeled antibody in the step (2) are paired antibodies, and form a double-antibody sandwich structure with the human eosinophil cationic protein and the human myeloperoxidase respectively; no cross reaction exists between the human eosinophil cationic protein pairing antibody and the human myeloperoxidase pairing antibody;
wherein, the preparation of the nitrocellulose NC film comprises the following steps: NC membrane coating, diluting the coated human eosinophil cationic protein and myeloperoxidase antibody to 0.5mg/ml, 1mg/ml or 1.5mg/ml by using 0.01M PBS (phosphate buffered saline) pH7.4, respectively diluting goat anti-mouse IgG to 1mg/ml or 2mg/ml, respectively coating by using a membrane spraying instrument according to 1.2ul/cm on the NC membrane by streaking, and drying the NC membrane for 2-5 hours at the temperature of 20-25 ℃ and the relative humidity of less than 30% in a drying room after coating;
(4) a sample pad tightly pressed against the nitrocellulose NC film;
wherein the kit is configured for detecting inflammatory factors in nasal secretions.
2. The human eosinophil cationic protein and myeloperoxidase assay kit according to claim 1, wherein the carrier is a microplate, magnetic particle, plastic tube or plastic bead.
3. The kit for detecting human eosinophil cationic protein and myeloperoxidase according to claim 1, wherein the enzyme for labeling in a) is alkaline phosphatase or horseradish peroxidase.
4. The human eosinophil cationic protein and myeloperoxidase detection kit according to claim 1, wherein the chromogenic substrate in the chemiluminescent immunoassay kit in a) is luminol, isoluminol, (adamantane) -1, 2-dioxirane, 3- (2 '-helical adamantane) -4-methoxy-4- (3' -phosphoryloxy) phenyl-1, 2-dioxirane, CSPD, or CDP-Star; the chromogenic substrate in the chemochromic immunoassay kit is 3,3', 5' -tetramethyl benzidine or diaminobenzidine.
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